Libertad García-Villada

Mutation of algae from sensitivity to resistance against environmental selective agents: the ecological genetics of Dictyosphaerium chlorelloides (Chlorophyceae) under lethal doses of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea herbicide

Abstract Residues contribute to water pollution and pose a challenge to microalgal populations because numerous contaminants are toxic to microalgae, even in the micromolar range. Adaptation of microalgae from herbicide sensitivity to herbicide resistance was analysed by an ecological genetic approach, using the unicellular alga Dictyosphaerium chlorelloides (Chlorophyceae) as an experimental model. A dose–effect study showed that the Malthusian parameter under conditions of r selection in an uncrowded environment and the carrying capacity under conditions of K selection in an crowded environment were both restricted even by low concentrations (< 1 μM) of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) herbicide. When a culture was treated with 50 μM DCMU, it cleared after a few days, as a result of destruction of sensitive cells by the herbicide. However, after further incubation for several days, the culture sometimes regained colour, owing to the growth of cell variants resistant to the herbicide. A fluctuation analysis was carried out to distinguish between (1) herbicide-resistant cells arising by direct and specific acquired adaptation in response to the herbicide and (2) herbicide-resistant cells arising by rare spontaneous mutations occurring randomly during replication of organisms prior to the incorporation of the herbicide. The fluctuation analysis unequivocally demonstrated that DCMU is not facilitating the development of DCMU-resistant cells; rather, we found that DCMU-resistant cells occur spontaneously by mutation in nonselective conditions prior to the incorporation of the herbicide (preselective mutations). The rate of spontaneous mutation from DCMU sensitivity to DCMU resistance was ∼ 2.2 × 10−6 mutants per cell division. Mutation was recurrent from a normal wild-type DCMU-sensitive allele to an DCMU-resistant allele, but such herbicide-resistant alleles were detrimental in terms of fitness in the absence of the herbicide. A competition experiment between wild-type DCMU-sensitive cells and DCMU-resistant mutants showed that, in small populations, the DCMU-resistant mutants are driven to extinction. The resistant variants are maintained in the absence of the herbicide as the result of a balance between new resistant cells arising by rare spontaneous mutation and resistant cells eliminated by natural selection. In our case, the average frequency of DCMU-resistant mutants in the absence of the DCMU is about five DCMU-resistant mutants per million cells. The results of our experimental model suggest that spontaneous mutation from herbicide sensitivity to herbicide resistance is sufficient in itself to assure the survival of microalgae populations in herbicide-contaminated environments when the population size is large.

ADAPTATION OF NEOSPORA CANINUM ISOLATES TO CELL-CULTURE CHANGES: AN ARGUMENT IN FAVOR OF ITS CLONAL POPULATION STRUCTURE

Neospora caninum, a recently recognized protozoan parasite of animals, is considered to be a major cause of bovine abortion worldwide. Although its life cycle is not completely known, recent studies suggest that the sexual stage occurs in dogs. The prevalence of sexual reproduction in N. caninum, however, is unknown. We investigated the ability of 3 N. caninum isolates (NC-1, NC-SweB1, and NC-Liverpool) to propagate asexually for approximately 250 parasite generations in a cell line in which they had not been cultured previously. The malthusian parameter of fitness was estimated for each isolate from 10 independent replicates of tachyzoites at the beginning as well as at the end of the experimental period. Derived and ancestral values for mean fitness were compared both within and among NC-1, NC-SweB1, and NC-Liverpool isolates. Results showed a significant increase in mean fitness for the 3 N. caninum isolates at the end of the experimental period. These findings indicate that N. caninum can adapt to new environmental conditions without the help of sexual recombination, supporting the idea that this parasite has, at least potentially, the capacity for maintaining clonal propagation in nature.

Evolution of microalgae in highly stressing environments: An experimental model analyzing the rapid adaptation of Dictyosphaerium chlorelloides (chlorophyceae) from sensitivity to resistance against 2,4,6-trinitrotoluene by rare preselective mutations

The increasing rates of global extinction due to human activities necessitate studies of the ability of organisms to adapt to the new environmental conditions resulting from human disturbances. We investigated the evolutionary adaptation of a microalga to sudden environmental change resulting from exposure to novel toxic chemical residues. A laboratory strain of Dictyosphaerium chlorelloides (Naum.) Kom. and Perm. (Chlorophyceae) was exposed to increasing concentrations of the modern contaminant 2,4,6‐trinitrotoluene (TNT). When algal cultures were exposed to 30 mg·L− 1 TNT, massive lysis of microalgal cells was observed. The key to understanding the evolution of microalgae in such a contaminated environment is to characterize the TNT‐resistant variants that appear after the massive lysis of the TNT‐sensitive cells. A fluctuation analysis demonstrated unequivocally that TNT did not facilitate the appearance of TNT‐resistant cells; rather it was found that TNT‐resistant cells appeared spontaneously by rare mutations under nonselective conditions, before exposure to TNT. The estimated mutation rate was 1.4 × 10−5 mutants per cell division. Isolated resistant mutants exhibited a diminished fitness in the absence of TNT. Moreover, the gross photosynthetic rate of TNT‐resistant mutants was significantly lower than that of wild‐type cells. Competition experiments between resistant mutants and wild‐type cells showed that in small populations, the resistant mutants were driven to extinction. The balance between mutation rate and the rate of selective elimination determines the occurrence of about 36 TNT‐resistant mutants per million cells in each generation. These scarce resistant mutants are the guarantee of potential for adaptation.

Short-term adaptation of microalgae in highly stressful environments : an experimental model analysing the resistance of Scenedesmus intermedius (Chlorophyceae) to the heavy metals mixture from the Aznalcollar mine spill

The toxic spill of acid wastes rich in heavy metals/metalloids (AWHM) from the Aznalcóllar mine in April 1998, threatening the Doñana National Park, is considered to be the worst environmental disaster related to acute pollution in Spanish history. The aim of this work was to study the adaptation of microalgae (which play an important role as primary producers) from AWHM sensitivity to AWHM resistance by using the alga Scenedesmus intermedius as an experimental model. The Malthusian parameter (m) and the carrying capacity (K) were reduced by mud and soil samples collected from the affected area. A dose–effect analysis showed that fitness progressively diminished with increasing sample concentration. A fluctuation analysis demonstrated that AWHM-resistant cells arose by rare spontaneous mutations that occurred randomly prior to the incorporation of the AWHM. The rate of spontaneous mutation from AWHM sensitivity to AWHM resistance was 2·12×10−5 mutants per cell division. A competition experiment between wild-type AWHM-sensitive cells and AWHM-resistant mutants showed that in small populations the AWHM-resistant mutants are driven to extinction in the absence of selection for AWHM resistance. The resistant phenotypes are maintained in the absence of AWHM as the result of a balance between spontaneous mutation and natural selection, so that about 43 AWHM-resistant mutants per million cells are present in the absence of AWHM. Our experimental model suggests that mutation is essential for adaptation of microalgal populations to environmental changes. Rare spontaneous pre-adaptive mutation is enough to ensure the survival of microalgal populations in contaminated environments when the population size is large enough.

Analysis of polygenic traits of Microcystis aeruginosa (Cyanobacteria) strains by Restricted Maximum Likelihood (REML) procedures: 2. Microcystin net production, photosynthesis and respiration

V. López-Rodas, E. Costas, E. Bañares, L. García-Villada, M. Altamirano, M. Rico, C. Salgado and A. Flores-Moya. 2006. Analysis of polygenic traits of Microcystis aeruginosa (Cyanobacteria) strains by restricted maximum likelihood (REML) procedures: 2. Microcystin net production, photosynthesis and respiration. Phycologia Phycologia 45: 243–248. DOI: 10.2216/04-31.1 Phenotypic expression of traits is the result of complex interactions between genotype and environment. A combination of experiments and statistics based on restricted maximum likelihood (REML) procedures was utilised for estimating the proportion of genetic variability in a series of phenotypic characters (microcystin net production, cell size, dark respiration rate, photosynthetic capacity, maximum quantum yield and growth rate), as well as their genetic and nongenetic correlations, measured on 18 Microcystis aeruginosa (Cyanobacteria) strains. Results indicated that the phenotypic variability found in these traits were principally due to genetic differences among strains. Thus, the estimated genetic variability ranged from ≈ 50% for dark respiration rate to > 90% in cell size or maximum quantum yield. These results support the conclusions that (1) genetic factors are the main cause of the spatial-temporal heterogeneity observed in quantum yield, respiration and toxin production, and (2) light harvesting and toxin production are not under intense natural selection, both having a low adaptive value. The high variance in toxin production resulting from genetic effects introduces a source of unpredictability in water-supply toxic early warnings. In addition, no significant genetic correlations were found between quantum yield and either maximal growth rate or mean cell diameter.